Abstract Silencing of tumor suppressor genes via aberrant epigenetic changes i.e., promoter hypermethylation and chromatin histone deacetylation are integral to malignant transformation. We have long focused on developing therapeutic strategies that target these aberrant epigenetic changes with the scope to restore normal patterns of hematopoietic cell proliferation, differentiation and apoptotsis in acute myeloid leukemia (AML). In the last funding cycle of this proposal, we were successful in identifying a biologically and clinically active, non-toxic low-dose of decitabine (20 mg/m2/day x 10 days every 4 weeks) that induced disease remission in untreated older (>60years) AML and high re-expression levels of the methylated ER gene, which in turn directly correlated with achievement of disease remission. We also produced intriguing preliminary data showing that decitabine modified expression of miRNAs, a novel class of non-coding genes that control translation of specific messenger RNA targets and in turn, levels of the corresponding encoded proteins (e.g., oncoproteins). Aberrant expression of miRNAs has been recently associated with cancer. miRNA 29, a microRNA that targets DNA methyltransferases (DNMT3a and 3 b), was found expressed at low levels in untreated patients and increased following decitabine treatment. Therefore, these data suggested that decitabine might interfere with aberrant DNMT activity also through re-expression of silenced miRNAs. Given the noteworthy interplay between DNA methylation and chromatin hypoacetylation in aberrant gene silencing in malignant cells, we combined decitabine with the histone deacetylase (HDAC) inhibitor valproic acid (VPA). Unfortunately, the addition of VPA was toxic and failed to provide any synergistic effect in terms of clinical response or gene re- expression. Finally, we obtained unreported evidence that the proteasome inhibitor bortezomib (Velcade", PS341) interfered with transactivation of the DNMT1 gene by reducing level and activity of the transcription factor Sp1. This resulted in reduction of DNMT1 levels, genomic DNA hypomethylation and methylated gene re-expression. Notably, bortezomib also induced expression of miRNA29b, which itself downregulated Sp1, and inhibited the activity of the transcription factor NF-B that is overexpressed in AML and cooperates with Sp1 in gene transactivation. Therefore, we hypothesize that bortezomib reduces aberrant DNMT activity by multiple mechanisms that target Sp1 via miRNA29 induction and/or NF-B inhibition. To capitalize on these novel discoveries, we propose here to conduct: 1) a phase II clinical trial of decitabine as a single agent in untreated elderly AML; 2) pharmacokinetic (PK) and pharmacodynamic (PD) correlative studies to define mechanistically, in vivo, the epigenetic activity of decitabine at the proposed dose and schedule; 3) a phase I dose-escalation trial of bortezomib and decitabine in combination in refractory/relapsed AML; 4) PK and PD correlative studies to define mechanistically, in vivo, the epigenetic activity of bortezomib. PUBLIC HEALTH RELEVANCE: Most of patients with acute myeloid leukemia (AML), a type of hematopoietic malignancies, have dismal outcome with current chemotherapy treatment, especially if older than 60 years, and therefore novel therapeutic approaches are highly needed. One of the mechanisms of development and maintenance of AML is represented by disruption of the function of genes important for normal activity of bone marrow and blood through methylation of their genomic DNA sequences. We are proposing here clinical trials with compounds (i.e., decitabine and bortezomib) that if used at specific doses and schedules, can decrease levels of DNA methylation, restore gene activity and result in disease remission. In addition to assessment of the clinical response to these compounds, we will analyze bone marrow and blood samples from treated patients to understand how bortezomib and/or decitabine reduce abnormal methylation in leukemia cells. This knowledge will be used to design more effective treatment for AML . [unreadable] [unreadable] [unreadable]